Prenylation of proteins by intermediates of the isoprenoid biosynthetic pathway represents a new class of post-translational modification (Glomset, J. A., Gelb, M. H., and Farnsworth, C. C. (1990). Trends Biochem. Sci. 15:139-142; Maltese, W. A. (1990). FASEB J. 4:3319-3328). This modification typically is required for the membrane localization and function of these proteins. Prenylated proteins-share characteristic C-terminal sequences including CaaX (C, Cys; a, usually aliphatic amino acid; X, another amino acid), XXCC, or XCXC. Three post-translational processing steps have been described for proteins having a C-terminal CaaX sequence: addition of either a 15 carbon (farnesyl) or 20 carbon (geranylgeranyl) isoprenoid to the Cys residue, proteolytic cleavage of the last 3 amino acids, and methylation of the new C-terminal carboxylate (Cox, A.D. and Der, C. J. (1992a). Critical Rev. Oncogenesis 3:365-400; Newman, C. M. H. and Magee, A. I. (1993). Biochim. Biophys. Acta 1155:79-96). Some proteins may also have a fourth modification: palmitoylation of one or two Cys residues N-terminal to the farnesylated Cys. Proteins terminating with a XXCC or XCXC motif are modified by geranylgeranylation on the Cys residues and do not require an endoproteolytic processing step. While some mammalian cell proteins terminating in XCXC are carboxymethylated, it is not clear whether carboxymethylation follows prenylation of proteins terminating with a XXCC motif (Clarke, S. (1992). Annu. Rev. Biochem. 61 :355-386). For all of the prenylated proteins, addition of the isoprenoid is the first step and is required for the subsequent steps (Cox, A. D. and Der, C. J. (1992a). Critical Rev. Oncogenesis 3:365-400; Cox, A. D. and Der, C. J. (1992b) Current Opinion Cell Biol. 4:1008-1016).
Three enzymes have been described that catalyze protein prenylation: farnesyl-protein transferase (FPTase), geranylgeranyl-protein transferase type I (GGPTase-I), and geranylgeranyl-protein transferase type-II (GGPTase-II, also called Rab GGPTase). These enzymes are found in both yeast and mammalian cells (Clarke, 1992; Schafer, W. R. and Rine, J. (1992) Annu. Rev. Genet. 30:209-237). FPTase and GGPTase-I are .alpha./.beta. heterodimeric enzymes that share a common .alpha. subunit; the .beta. subunits are distinct but share approximately 30% amino acid similarity (Brown, M. S. and Goldstein, J. L. (1993). Nature 366: 14-15; Zhang, F. L.; Diehi, R. E., Kohl, N. E., Gibbs, J. B., Giros, B., Casey, P. J., and Omer, C. A. (1994). J. Biol. Chem. 269:3175-3180). GGPTase-II has different a and 13 subunits and complexes with a third component (REP, Rab Escort Protein) that presents the protein substrate to the .alpha./.beta. catalytic subunits. Each of these enzymes selectively uses famesyl diphosphate or geranylgeranyl diphosphate as the isoprenoid donor and selectively recognizes the protein substrate. FPTase farnesylates CaaX-containing proteins that end with Ser, Met, Cys, GIn, Phe or Ala. GGPTase-I geranylgeranylates CaaX-containing proteins that end with Leu or Phe. For FPTase and GGPTase-I, CaaX tetrapeptides comprise the minimum region required for interaction of the protein substrate with the enzyme. GGPTase-II modifies XXCC and XCXC proteins; the interaction between GGPTase-II and its protein substrates is more complex, requiring protein sequences in addition to the C-terminal amino acids for recognition. The enzymological characterization of these three enzymes has demonstrated that it is possible to selectively inhibit one with little inhibitory effect on the others (Moores, S. L., Schaber, M. D., Mosser, S. D., Rands, E., O'Hara, M. B., Garsky, V. M., Marshall, M. S., Pompliano, D. L., and Gibbs, J. B., J. Biol. Chem., 266:17438 (1991)).
The characterization of protein prenylation biology and enzymology has opened new areas for the development of inhibitors which can modify physiological processes. The prenylation reactions have been shown genetically to be essential for the function of a variety of proteins (Clarke, 1992; Cox and Der, 1992a; Gibbs, J. B. (1991). Cell 65:1-4; Newman and Magee, 1993; Schafer and Rine, 1992). This requirement often is demonstrated by mutating the CaaX Cys acceptors so that the proteins can no longer be prenylated. The resulting proteins are devoid of their central biological activity. These studies provide a genetic "proof of principle" indicating that inhibitors of prenylation can alter the physiological responses regulated by prenylated proteins.
The Ras gene is found activated in many human cancers, including colorectal carcinoma, exocrine pancreatic carcinoma, and myeloid leukemias. Biological and biochemical studies of Ras action indicate that Ras functions like a G-regulatory protein, since Ras must be localized in the plasma membrane and must bind with GTP in order to transform cells (Gibbs, J. et al., Microbiol. Rev. 53:171-286 (1989). Forms of Ras in cancer cells have mutations that distinguish the protein from Ras in normal cells.
Famesylation of Ras by the isoprenoid famesyl pyrophosphate (FPP) occurs in vivo on Cys to form a thioether linkage (Hancock et al., Cell 57:1167 (1989); Casey et al., Proc. Natl. Acad. Sci. USA 86:8323 (1989)). In addition, Ha-Ras and N-Ras are palmitoylated via formation of a thioester on a Cys residue near a C-terminal Cys famesyl acceptor (Gutierrez et al., EMBO J. 8:1093-1098 (1989); Hancock et al., Cell 57:1167-1177 (1989)). Ki-Ras lacks the palmitate acceptor Cys. The last 3 amino acids at the Ras C-terminal end are removed proteolytically, and methyl esterification occurs at the new C-terminus (Hancock et al., ibid). Fungal mating factor and mammalian nuclear lamins undergo identical modification steps (Anderegg et al., J. Biol. Chem. 263:18236 (1988); Famsworth et al., J. Biol. Chem. 264:20422 (1989)).
Inhibition of Ras famesylation in vivo has been demonstrated with Lovastatin (Merck & Co., Rahway, N.J.) and compactin (Hancock et al., ibid; Casey et al., ibid; Schafer et al., Science 245:379 (1989)). These drugs inhibit HMG-CoA reductase, the rate limiting enzyme for the production of polyisoprenoids and the famesyl pyrophosphate precursor. It has been shown that a famesyl-protein transferase using famesyl pyrophosphate as a precursor is responsible for Ras famesylation. (Reiss et al., Cell, 62:81-88 (1990); Schaber et al., J. Biol. Chem., 265:14701-14704 (1990); Schafer et al., Science, 249:1133-1139 (1990); Manne et al., Proc. Natl. Acad. Sci. USA, 87:7541-7545 (1990)).
Famesyl-protein transferase activity may be reduced or completely inhibited by adjusting the compound dose. Reduction of famesyl-protein transferase enzyme activity by adjusting the compound dose would be useful for avoiding possible undesirable side effects resulting from interference with other metabolic processes which utilize the enzyme.
These compounds and their analogs are inhibitors of famesyl-protein transferase. Famesyl-protein transferase utilizes famesyl pyrophosphate to covalently modify the Cys thiol group of the Ras CAAX box with a famesyl group. Inhibition of famesyl pyrophosphate biosynthesis by inhibiting HMG-CoA reductase blocks Ras membrane localization in vivo and inhibits Ras function. Inhibition of famesyl-protein transferase is more specific and is attended by fewer side effects than is the case for a general inhibitor of isoprene biosynthesis.
Previously, it has been demonstrated that tetrapeptides containing cysteine as an amino terminal residue with CAAX sequence inhibit Ras famesylation (Schaber et al., ibid; Reiss et al., ibid; Reiss et al., PNAS, 88:732-736 (1991)). Such inhibitors may inhibit while serving as alternate substrates for the Ras famesyl-transferase enzyme, or may be purely competitive inhibitors (U.S. Pat. No. 5,141,851, University of Texas).
Recently, it has been demonstrated that certain inhibitors of farnesyl-protein transferase selectively block the processing of Ras oncoprotein intracellularly (N. E. Kohl et al., Science, 260:1934-1937 (1993) and G. L. James et al., Science, 260:1937-1942 (1993).
Certain non-peptide analogs of fameysl diphosphate (FFP) also inhibit famesyl-protein transferase (Anthony et al., U.S. Pat. No. 5,298,655, (Mar. 29, 1994)). It has also been noted that some non-peptidal analogs of FPP are selective inhibitors of famesyl-protein transferase (J. B. Gibbs et al., J. Biol. Chem. (1993) 268:7617).
Protein geranylgeranyltransferase type-I (GGTase-I) transfers a geranylgeranyl group from the prenyl donor geranylgeranyl diphosphate to the cysteine residue of substrate proteins containing a C-terminal CAAX-motif in which the "X" residue is leucine or phenylalanine (Clark, 1992; Newman and Magee, 1993). Known targets of GGTase-I include the gamma subunits of brain heterotrimeric G proteins and Ras-related small GTP-binding proteins such as RhoA, RhoB, RhoC, CDC42Hs, Rac1, Rac2, Rap1A and Rap1B (Newman and Magee, 1993; Cox and Der, 1992a). The proteins RhoA, RhoB, RhoC, Rac1, Rac2 and CDC42Hs have roles in the regulation of cell shape (Ridley, A. J. and Hall, A. (1992). Cell 70:389-399; Ridley, A. J., Paterson, H. F., Johnston, C. L., Keikmann, D., and Hall, A. (1992). Cell 70:401-410; Bokoch, G. M. and Der, C. J. (1993). FASEB J. 7:750-759). Rac and Rap proteins have roles in neutrophil activation s (Bokoch and Der, 1993 ).
Activation of growth factor function and Ras function can cause tumor formation. Recently, it was demonstrated that the Rho and Rac proteins transmit intracellular signals initiated by growth factors and by Ras protein (Prendergast, G. C. and Gibbs, J. B. (1993). Adv. Cancer Res. 62:19-64; Ridley and Hall, 1992; Ridley et al., 1992). Specifically, experiments demonstrated that the function of Rho and Rac proteins was required by Ras and growth factors to change cell shape, a biological parameter indicative of cellular transformation and cancer. Since Rho and Rac proteins require geranylgeranylation for function, an inhibitor of GGPTase-I would block the functions of these proteins and be useful as an anticancer agent.
Neutrophil activation is part of the body's inflammatory response. (Haslett, C. et al., Cur. Opinion Immunology, 2:10-18 (1989) Geranylgeranylated Rac and Rap proteins are required for this effect (Bokoch and Der (1993); Abo, A. et al., Nature, 353:668-670 (1991); Knaus, U. G. et al., Science, 254:1512-1515 (1991); Eklund, E. A. et al., J. Biol. Chem. 266:13964-13970 (1991); Quinn, M.T. et al., Nature, 342:198-200 (1989)), so an inhibitor of GGPTase-I will have anti-inflammatory activity.